The rapidly growing world population and the high rate of unintended pregnancies make contraception a need and a priority for any public health program. While several contraceptive methods, with varying efficacy are currently available for women, a more comprehensive approach to birth control requires extending contraception to males. However, a safe, effective and reversible contraceptive for men is still unavailable. An attractive approach to develop male contraceptives consists in targeting proteins that are specifically expressed in sperm and are required for sperm fertility. We have shown that Na,K-ATPase ?4 (NKA?4), a plasma membrane ion transporter which exchanges cytoplasmic Na+ for extracellular K+, is a validated target for male contraception. NKA?4 is uniquely expressed in testis male germ cells after meiosis, is particularly abundant in the sperm flagellum, and is critical for sperm function. Deletion of NAK?4 in mice results in complete sterility of only the male but not the female animals. NKA?4 is essential for sperm motility and sperm capacitation. Its activity maintains sperm intracellular Na+ levels ([Na+]i) and several vital sperm parameters, including membrane potential (Vm), intracellular Ca+2 ([Ca2+]i) and pH. From a biochemical standpoint, NKA?4 has a particularly high affinity for ouabain, the specific inhibitor of Na,K-ATPase. We took advantage of this property to specifically target NKA?4 and block its function to achieve male infertility. We synthesized a series of small molecule compounds, which can selectively bind to the high ouabain affinity site of NKA?4. Some of these compounds inhibit NKA?4 and affect sperm motility both in vitro and after administration to mice. This provides strong evidence for the suitability of NKA?4 as a pharmacological target and our compounds as agents that can be used for the control of male fertility. However, before NKA?4 inhibitors can be moved forward into their application as male contraceptives, it is necessary that their efficacy, drug-target interaction, biomarkers for their in vitro and vivo specificity, side effects, mechanisms of action and pharmacokinetic parameters are identified and optimized for future clinical use. We will test this in two aims. In specific aim 1, we will develop compounds with the capacity to selectively inhibit NKA?4 and block sperm function, which will be ready for testing in mice. Then, during specific aim 2, we will perform studies to obtain preclinical validation to advance the NKA?4 inhibitors as male contraceptives. A series of rigorous approaches from the medicinal chemistry and biological areas will be used to identify the compounds which will have the characteristics that will be necessary for male contraception. This research will be essential to fulfill the highly desired unmet goal of obtaining a non-hormonal pharmacological agent that could be used as an oral, reversible agent for the control of male fertility.